Genomic surveillance of coxsackievirus A10 reveals genetic features and recent appearance of genogroup D in Shanghai,China,2016–2020

Coxsackievirus A10(CVA10)is one of the major causative agents of hand,foot and mouth disease(HFMD).To investigate the epidemiological characteristics as well as genetic features of CVA10 currently circulating in Shanghai,China,we collected a total of 9,952 sporadic HFMD cases from January 2016 to December 2020.In the past five years,CVA10 was the fourth prevalent causatives associated with HFMD in Shanghai and the overall positive rate was 2.78%.The annual distribution experienced significant fluctuations over the past five years.In addition to entire VP1 sequencing,complete genome sequencing and recombination analysis of CVA10 isolates in Shanghai were further performed.A total of 64 near complete genomes and 11 entire VP1 sequences in this study combined with reference sequences publicly available were integrated into phylogenetic analysis.The CVA10sequences in this study mainly belonged to genogroup C and presented 91%-100%nucleotide identity with other Chinese isolates based on VP1 region.For the first time,our study reported the appearance of CVA10 genogroup D in Chinese mainland,which had led to large-scale outbreaks in Europe previously.The recombination analysis showed the recombination break point located between 5,100 nt and 6,700 nt,which suggesting intertypic recombination with CVA16 genogroup D.To conclusion,CVA10 genogroup C was the predominant genogroup in Shanghai during 2016-2020.CVA10 recombinant genogroup D was firstly reported in circulating in Chinese mainland.Continuous surveillance is needed to better understand the evolution relationships and transmission pathways of CVA10 to help to guide disease control and prevention.

The Establishment of Infectious Clone and Single Round Infectious Particles for Coxsackievirus A10

Coxsackievirus A10(CVA10)is one of the major etiological agents of hand,foot,and mouth disease.There are no vaccine and antiviral drugs for controlling CVA10 infection.Reverse genetic tools for CVA10 will benefit its mechanistic study and development of vaccines and antivirals.Here,two infectious clones for the prototype and a Myc-tagged CVA10 were constructed.Viable CVA10 viruses were harvested by transfecting the viral m RNA into human rhabdomyosarcoma(RD)cells.Rescued CVA10 was further confirmed by next generation sequencing and characterized experimentally.We also constructed the vectors for CVA10 subgenomic replicon with luciferase reporter and viral capsid with EGFP reporter,respectively.Co-transfection of the viral replicon RNA and capsid expresser in human embryonic kidney 293 T(HEK293 T)cells led to the production of single round infectious particles(SRIPs).Based on CVA10 replicon RNA,SRIPs with either the enterovirus A71(EVA71)capsid or the CVA10 capsid were generated.Infection by EVA71 SRIPs required SCARB2,while CVA10 SRIPs did not.Finally,we showed great improvement of the replicon activity and SRIPs production by insertion of a cis-active hammerhead ribozyme(HHRib)before the 50-untranslated region(UTR).In summary,reverse genetic tools for prototype strain of CVA10,including both the infectious clone and the SRIPs system,were successfully established.These tools will facilitate the basic and translational study of CVA10.

Pathogenic analysis of coxsackievirus A10 in rhesus macaques

Coxsackievirus A10(CV-A10)is one of the etiological agents associated with hand,foot and mouth disease(HFMD)and also causes a variety of illnesses in humans,including pneumonia,and myocarditis.Different people,particularly young children,may have different immunological responses to infection.Current CV-A10 infection animal models provide only a rudimentary understanding of the pathogenesis and effects of this virus.The characteristics of CV-A10 infection,replication,and shedding in humans remain unknown.In this study,rhesus macaques were infected by CV-A10 via respiratory or digestive route to mimic the HFMD in humans.The clinical symptoms,viral shedding,inflammatory response and pathologic changes were investigated in acute infection(1–11 day post infection)and recovery period(12–180 day post infection).All infected rhesus macaques during acute infection showed obvious viremia and clinical symptoms which were comparable to those observed in humans.Substantial inflammatory pathological damages were observed in multi-organs,including the lung,heart,liver,and kidney.During the acute period,all rhesus macaques displayed clinical signs,viral shedding,normalization of serum cytokines,and increased serum neutralizing antibodies,whereas inflammatory factors caused some animals to develop severe hyperglycemia during the recovery period.In addition,there were no significant differences between respiratory and digestive tract infected animals.Overall,all data presented suggest that the rhesus macaques provide the first non-human primate animal model for investigating CV-A10 pathophysiology and assessing the development of potential human therapies.